This grant will focus on the the expression and function during development of specific components of the mammalian insulin-like growth factor (IGF) system, which is known to effect fetal growth in vivo. The system includes two peptide growth factors (IGF-I and IGF-II), at least two IGF receptors, and a family of six recently-cloned IGF binding protein genes (IGFBPs). We will complete ongoing studies on the expression of one IGFBP, IGFBP-2, which is developmentally regulated and expressed in the embryonic ectoderm of early post-implantation rat embryos; at later ages, IGFBP-2 expression is high primarily in ectodermal regions of morphogenetic interest including the ectodermal placodes, the apical ectodermal ridge of the limb bud, and ventricular cells of the CNS. We will extend analysis of IGFBP-2 expression to both earlier and later ages. The experiments on earlier ages will determine the relationship between onset of IGFBP-2 expression and appearance of the inner cell mass; those at later ages will determine whether IGFBP-2 expression occurs in progenitors of neurons and glia. We will localize this RGD-containing protein immunocytochemically to determine the relationship between sites of IGFBP-2 synthesis and possible sites of IGFBP-2 action. We will then determine the function of IGFBP-2 during development using a gene targeting strategy. We will use a mouse genomic clone we have already isolated to construct a targeting vector containing IGFBP-2 genomic sequence and selectable neomycin and herpes simplex thymidine kinase genes. We will replace one IGFBP-2 allele in embryonic stem (ES) cells by introducing the targeting vector into these cells by electroporation, selecting with gancyclovir and neomycin, and identifying homologous recombinants among the surviving colonies by PCR and Southern blotting. ES cells with an altered IGFBP-2 allele will be introduced into mice by blastocyst injection and the phenotypes of hetero- and homozygote offspring from germ line chimeras will be assessed. To complement these studies, we will determine the pattern of expression of newly-isolated IGFBP genes during fetal development, and complete our ongoing studies of IGF-I expression (which appears to identify limb bud mesoderm) and the type 1 IGF receptor. Taken together, these studies will clarify the role of the IGF system in prenatal development and likely will provide information relevant to prenatal growth and possibly to specific birth defects.